Method for preparing modified subtilisins

ABSTRACT

There are described methods for making mutant subtilisins, the methods comprising obtaining a DNA fragment from a Bacillus subtilisin and introducing a mutation into the fragment by substituting at least one amino acid, transforming a suitable host cell with the mutated DNA, recovering a mutant subtilisin and screening the mutant subtilisin for certain altered enzymatic properties.

This application is a continuation of U.S. patent application Ser. No.07/091,235, filed Aug. 31, 1987 (now abandoned) which is a divisionalapplication of U.S. patent application Ser. No. 06/614,612, filed May29, 1984, issued as U.S. Pat. No. 4,760,025 on Jul. 26, 1988.

BACKGROUND

This invention relates to the production and manipulation of proteinsusing recombinant techniques in suitable hosts. More specifically, theinvention relates to the production of procaryotic proteases such assubtiltsin and neutral protease using recombinant microbial host cells,to the synthesis of heterologous proteins by microbial hosts, and to thedirected mutagenesis of enzymes in order to modify the characteristicsthereof.

Various bacteria are known to secrete proteases at some stage in theirlife cycles. Bacillus species produce two major extracellular proteases,a neutral protease (a metalloprotease inhibited by EDTA) and an alkalineprotease (or subtilisin, a serine endoprotease). Both generally areproduced in greatest quantity after the exponential growth phase, whenthe culture enters stationary phase and begins the process ofsporulation. The physiological role of these two proteases is not clear.They have been postulated to play a role in sporulation (J. Hoch, 1976,"Adv. Genet." 18:69-98; P. Piggot et al., 1976, "Bact. Rev." 40:908-962;and F. Priest, 1977, "Bact. Rev." 41:711-753), to be involved in theregulation of cell wall turnover (L. Jolliffe et al., 1980, "J. Bact."141:1199-1208), and to be scavenger enzymes (Priest, Id.). Theregulation of expression of the protease genes is complex. They appearto be coordinately regulated in concert with sporulation, since mutantsblocked in the early stages of sporulation exhibit reduced levels ofboth the alkaline and neutral protease. Additionally, a number ofpleiotropic mutations exist which affect the level of expression ofproteases and other secreted gene products, such as amylase andlevansucrase (Priest, Id.).

Subtilisin has found considerable utility in industrial and commercialapplications (see U.S. Pat. No. 3,623,957 and J. Millet, 1970, "J. Appl.Bact." 33:207). For example, subtilisins and other proteases arecommonly used in detergents to enable removal of protein-based stains.They also are used in food processing to accommodate the proteinaceoussubstances present in the food preparations to their desired impact onthe composition.

Classical mutagenesis of bacteria with agents such as radiation orchemicals has produced a plethora of mutant strains exhibiting differentproperties with respect to the growth phase at which protease excretionoccurs as well as the timing and activity levels of excreted protease.These strains, however, do not approach the ultimate potential of theorganisms because the mutagenic process is essentially random, withtedious selection and screening required to identify organisms whicheven approach the desired characteristics. Further, these mutants arecapable of reversion to the parent or wild-type strain. In such eventthe desirable property is lost. The probability of reversion is unknownwhen dealing with random mutagenesis since the type and site of mutationis unknown or poorly characterized. This introduces considerableuncertainty into the industrial process which is based on theenzyme-synthesizing bacterium. Finally, classical mutagenesis frequentlycouples a desirable phenotype, e.g., low protease levels, with anundesirable character such as excessive premature cell lysis.

Special problems exist with respect to the proteases which are excretedby Bacillus. For one thing, since at least two such proteases exist,screening for the loss of only one is difficult. Additionally, the largenumber of pleiotropic mutations affecting both sporulation and proteaseproduction make the isolation of true protease mutations difficult.

Temperature sensitive mutants of the neutral protease gene have beenobtained by conventional mutagenic techniques, and were used to map theposition of the regulatory and structural gene in the Bacillus subtillischromosome (H. Uehara et al., 1979, "J. Bact." 139:583-590).Additionally, a presumed nonsense mutation of the alkaline protease genehas been reported (C. Roitsch et al., 1983, "J. Bact." 155:145-152).

Bacillus temperature sensitive mutants have been isolated that produceinactive serine protease or greatly reduced levels of serine protease.These mutants, however, are asporogenous and show a reversion frequencyto the wild-type of about from 10⁻⁷ to 10⁻⁸ (F. Priest, Id. p. 719).These mutants are unsatisfactory for the recombinant production ofheterologous proteins because asporogenous mutants tend to lyse duringearlier stages of their growth cycle in minimal medium than dosporogenic mutants, thereby prematurely releasing cellular contents(including intracellular proteases) into the culture supernatant. Thepossibility of reversion also is undesirable since wild-type revertantswill contaminate the culture supernatant with excreted proteases.

Bacillus sp. have been proposed for the expression of heterologousproteins, but the presence of excreted proteases and the potentialresulting hydrolysis of the desired product has retarded the commercialacceptance of Bacillus as a host for the expression of heterologousproteins. Bacillus megaterium mutants have been disclosed that arecapable of sporulation and which do not express a sporulation-associatedprotease during growth phases. However, the assay employed did notexclude the presence of other proteases, and the protease in question isexpressed during the sporulation phase (C. Loshon et al., 1982, "J.Bact." 150:303-311). This, of course, is the point at which heterologousprotein would have accumulated in the culture and be vulnerable. It isan objective herein to construct a Bacillus strain that is substantiallyfree of extracellular neutral and alkaline protease during all phases ofits growth cycle and which exhibits substantially normal sporulationcharacteristics. A need exists for non-revertible, otherwise normalprotease deficient organisms that can then be transformed with high copynumber plasmids for the expression of heterologous or homologousproteins.

Enzymes having characteristics which vary from available stock arerequired. In particular, enzymes having enhanced oxidation stabilitywill be useful in extending the shelf life and bleach compatibility ofproteases used in laundry products. Similarly, reduced oxidationstability would be useful in industrial processes that require the rapidand efficient quenching of enzymatic activity.

Modifying the pH-activity profiles of an enzyme would be useful inmaking the enzymes more efficient in a wide variety of processes, e.g.broadening the pH-activity profile of a protease would produce an enzymemore suitable for both alkaline and neutral laundry products. Narrowingthe profile, particularly when combined with tailored substratespecificity, would make enzymes in a mixture more compatible, as will befurther described herein.

Mutations of procaryotic carbonyl hydrolases (principally proteases butincluding lipases) will facilitate preparation of a variety of differenthydrolases, particularly those having other modified properties such asKm, Kcat, Km/Kcat ratio and substrate specificity. These enzymes canthen be tailored for the particular substrate which is anticipated to bepresent, for example in the preparation of peptides or for hydrolyticprocesses such as laundry uses.

Chemical modification of enzymes is known. For example, see I. Svendsen,1976, "Carlsberg Res. Commun." 41 (5): 237-291. These methods, however,suffer from the disadvantages of being dependent upon the presence ofconvenient amino acid residues, are frequently nonspecific in that theymodify all accessible residues with common side chains, and are notcapable of reaching inaccessible amino acid residues without furtherprocessing, e.g. denaturation, that is generally not completelyreversible in reinstituting activity. To the extent that such methodshave the objective of replacing one amino acid residue side chain foranother side chain or equivalent functionality, then mutagenesispromises to supplant such methods.

Predetermined, site-directed mutagenesis of tRNA synthetase in which acys residue is converted to serine has been reported (G. Winter et al.,1982, "Nature" 299:756-758; A. Wilkinson et al., 1984, "Nature"307:187-188). This method is not practical for large scale mutagenesis.It is an object herein to provide a convenient and rapid method formutating DNA by saturation mutagenesis.

Summary

A method for producing procaryotic carbonyl hydrolase such as subtilisinand neutral protease in recombinant host cells is described in whichexpression vectors containing sequences which encode desired subtilisinor neutral protease, including the pro, pre, or prepro forms of theseenzymes, are used to transform hosts, the host cultured and desiredenzymes recovered. The coding sequence may correspond exactly to onefound in nature, or may contain modifications which confer desirableproperties on the protein that is produced, as is further describedbelow.

The novel strains then are transformed with at least one DNA moietyencoding a polypeptide not otherwise expressed in the host strain, thetransformed strains cultured and the polypeptide recovered from theculture. Ordinarily, the DNA moiety is a directed mutant of a hostBacillus gene, although it may be DNA encoding a eucaryotic (yeast ormammalian) protein. The novel strains also serve as hosts for proteinexpressed from a bacterial gene derived from sources other than the hostgenome, or for vectors expressing these heterologous genes, orhomologous genes from the host genome. In the latter event enzymes suchas amylase are obtained free of neutral protease or subtilisin. Inaddition, it is now possible to obtain neutral protease in culture whichis free of enzymatically active subtilisin, and vice-versa.

One may, by splicing the cloned genes for procaryotic carbonyl hydrolaseinto a high copy number plasmid, synthesize the enzymes in enhancedyield compared to the parental organisms. Also disclosed are modifiedforms of such hydrolases, including the pro and prepro zymogen forms ofthe enzymes, the pre forms, and directed mutations thereof.

A convenient method is provided for saturation mutagenesis, therebyenabling the rapid and efficient generation of a plurality of mutationsat any one site within the coding region of a protein, comprising;

(a) obtaining a DNA moiety encoding at least a portion of said precursorprotein;

(b) identifying a region within the moiety;

(c) substituting nucleotides for those already existing within theregion in order to create at least one restriction enzyme site unique tothe moiety, whereby unique restriction sites 5' and 3' to the identifiedregion are made available such that neither alters the amino acids codedfor by the region as expressed;

(d) synthesizing a plurality of oligonucleotides, the 5' and 3' ends ofwhich each contain sequences capable of annealing to the restrictionenzyme sites introduced in step (c) and which, when ligated to themoiety, are expressed as substitutions, deletions and/or insertions ofat least one amino acid in or into said precursor protein;

(e) digesting the moiety of step (c) with restriction enzymes capable ofcleaving the unique sites; and

(f) ligating each of the oligonucleotides of step (d) into the digestedmoiety of step (e) whereby a plurality of mutant DNA moleties areobtained.

By the foregoing method or others known in the art, a mutation isintroduced into isolated DNA encoding a procaryotic carbonyl hydrolasewhich, upon expression of the DNA, results in the substitution, deletionor insertion of at least one amino acid at a predetermined site in thehydrolase. This method is useful in creating mutants of wild typeproteins (where the "precursor" protein is the wild type) or revertingmutants to the wild type (where the "precursor" is the mutant.

Mutant enzymes are recovered which exhibit oxidative stability and/orpH-activity profiles which differ from the precursor enzymes.Procaryotic carbonyl hydrolases having varied Km, Kcat, Kcat/Km ratioand substrate specificity also are provided herein.

The mutant enzymes obtained by the methods herein are combined in knownfashion with surfactants or detergents to produce novel compositionsuseful in the laundry or other cleaning arts.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1A, the entire functional sequence for B. amyloliquefaciens,including the promoter and ribosome binding site, are present on a 1.5kb fragment of the B. amyloliquefaciens genome.

FIG. 1B shows the nucleotide sequence of the coding strand, correlatedwith the amino acid sequence of the protein. Promoter (p) ribosomebinding site (rbs) and termination (term) regions of the DNA sequenceare also shown.

FIG. 2 shows the results of replica nitrocellulose filters of purifiedpositive clones probed with Pool 1 (Panel A) and Pool 2 (Panel B)respectively.

FIG. 3 shows the restriction analysis of the subtilisin expressionplasmid (pS4). pBS42 vector sequences (4.5 kb) are shown in solid whilethe insert sequence (4.4 kb) is shown dashed.

FIG. 4 shows the results of SDS-PAGE performed on supernatants fromcultures transformed with pBS42 and pS4.

FIG. 5 shows the construction of the shuttle vector pBS42.

FIG. 6 shows a restriction map for a sequence including the B. subtillssubtilisin gene.

FIG. 7 (7A-1, 7A-2, 7A-3) is the sequence of a functional B. subtilissubtilisin gene.

FIG. 8 demonstrates a construction method for obtaining a deletionmutant of a B. subtills subtilisin gene.

FIG. 9 discloses the restriction map for a B. subtills neutral proteasegene.

FIG. 10A-1, 10A-2 and 10A-3 are the nucleotide sequence for a B.subtilis neutral protease gene.

FIG. 11 demonstrates the construction of a vector containing a B.subtilis neutral protease gene.

FIGS. 12, 13 and 16 disclose embodiments of the mutagenesis techniqueprovided herein.

FIG. 14 shows the enhanced oxidation stability of a subtilisin mutant.

FIG. 15 demonstrates a change in the pH-activity profile of a subtilisinmutant when compared to the wild type enzyme.

DETAILED DESCRIPTION

Procaryotic carbonyl hydrolases are enzymes which hydrolyze compoundscontaining ##STR1## bonds in which X is oxygen or nitrogen. Theyprincipally include hydrolases, e.g. lipases and peptide hydrolases,e.g. subtilisins or metalloproteases. Peptide hydrolases includeα-aminoacylpeptide hydrolase, peptidylamino-acid hydrolase, acylaminohydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiolproteinase, carboxylproteinase and metalloproteinase. Serine, metallo,thiol and acid proteases are included, as well as endo andexo-proteases.

Subtilisins are serine proteinases which generally act to cleaveinternal peptide bonds of proteins or peptides. Metalloproteases areexo- or endoproteases which require a metal ion cofactor for activity.

A number of naturally occurring mutants of subtilisin or neutralprotease exist, and all may be employed with equal effect herein assources for starting genetic material.

These enzymes and their genes may be obtained from many procaryoticorganisms. Suitable examples include gram negative organisms such as E.coli or pseudomonas and gram positive bacteria such as micrococcus orbacillus.

The genes encoding the carbonyl hydrolase may be obtained in accord withthe general method herein. As will be seen from the examples, thiscomprises synthesizing labelled probes having putative sequencesencoding regions of the hydrolase of interest, preparing genomiclibraries from organisims expressing the hydrolase, and screening thelibraries for the gene of interest by hybridization to the probes.Positively hybridizing clones are then mapped and sequenced. The clonedgenes are ligated into an expression vector (which also may be thecloning vector) with requisite regions for replication in the host, theplasmid transfected into a host for enzyme synthesis and the recombinanthost cells cultured under conditions favoring enzyme synthesis, usuallyselection pressure such as is supplied by the presence of an antibiotic,the resistance to which is encoded by the vector. Culture under theseconditions results in enzyme yields multifold greater than the wild typeenzyme synthesis of the parent organism, even if it is the parentorganism that is transformed.

"Expression vector" refers to a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of said DNA in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites, and sequences which controltermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, "plasmid" and"vector" are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

"Recombinant host cells" refers to cells which have been transformed ortransfected with vectors constructed using recombinant DNA techniques.As relevant to the present invention, recombinant host cells are thosewhich produce procaryotic carbonyl hydrolases in its various forms byvirtue of having been transformed with expression vectors encoding theseproteins. The recombinant host cells may or may not have produced a formof carbonyl hydrolase prior to transformation.

"Operably linked" when describing the relationship between two DNAregions simply means that they are functionally related to each other.For example, a presequence is operably linked to a peptide if itfunctions as a signal sequence, participating in the secretion of themature form of the protein most probably involving cleavage of thesignal sequence. A promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; a ribosome binding siteis operably linked to a coding sequence if it is positioned so as topermit translation.

"Prohydrolase" refers to a hydrolase which contains additionalN-terminal amino acid residues which render the enzyme inactive but,when removed, yield an enzyme. Many proteolytic enzymes are found innature as translational proenzyme products and, in the absence ofpost-translational products, are expressed in this fashion.

"Presequence" refers to a signal sequence of amino acids bound to theN-terminal portion of the hydrolase which may participate in thesecretion of the hydrolase. Presequences also may be modified in thesame fashion as is described here, including the introduction ofpredetermined mutations. When bound to a hydrolase, the subject proteinbecomes a "prehydrolase". Accordingly, relevant prehydrolase for thepurposes herein are presubtilisin and preprosubtilisin. Prehydrolasesare produced by deleting the "pro" sequence (or at least that portion ofthe pro sequence that maintains the enzyme in its inactive state) from aprepro coding region, and then expressing the prehydrolase. In this waythe organism excretes the active rather than proenzyme.

The cloned carbonyl hydrolase is used to transform a host cell in orderto express the hydrolase. This will be of interest where the hydrolasehas commercial use in its unmodified form, as for example subtilisin inlaundry products as noted above. In the preferred embodiment thehydrolase gene is ligated into a high copy number plasmid. This plasmidreplicates in hosts in the sense that it contains the well-knownelements necessary for plasmid replication: a promoter operably linkedto the gene in question (which may be supplied as the gene's ownhomologous promotor if it is recognized, i.e., transcribed, by thehost), a transcription termination and polyadenylation region (necessaryfor stability of the mRNA transcribed by the host from the hydrolasegene) which is exogenous or is supplied by the endogenous terminatorregion of the hydrolase gene and, desirably, a selection gene such as anantibiotic resistance gene that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antibiotic-containing media.High copy number plasmids also contain an origin of replication for thehost, thereby enabling large numbers of plasmids to be generated in thecytoplasm without chromosonal limitations. However, it is within thescope herein to integrate multiple copies of the hydrolase gene intohost genome. This is facilitated by bacterial strains which areparticularly susceptible to homologous recombination. The resulting hostcells are termed recombinant host cells.

Once the carbonyl hydrolase gene has been cloned, a number ofmodifications are undertaken to enhance the use of the gene beyondsynthesis of the wild type or precursor enzyme. A precursor enzyme isthe enzyme prior to its modification as described in this application.Usually the precursor is the enzyme as expressed by the organism whichdonated the DNA modified in accord herewith. The term "precursor" is tobe understood as not implying that the product enzyme was the result ofmanipulation of the precursor enzyme per se.

In the first of these modifications, the gene may be deleted from arecombination positive (rec⁺) organism containing a homologous gene.This is accomplished by recombination of an in vitro deletion mutationof the cloned gene with the genome of the organism. Many strains oforganisms such as E. coli and Bacillus are known to be capable ofrecombination. All that is needed is for regions of the residual DNAfrom the deletion mutant to recombine with homologous regions of thecandidate host. The deletion may be within the coding region (leavingenzymatically inactive polypeptides) or include the entire coding regionas long as homologous flanking regions (such as promoters or terminationregions) exist in the host. Acceptability of the host for recombinationdeletion mutants is simply determined by screening for the deletion ofthe transformed phenotype. This is most readily accomplished in the caseof carbonyl hydrolase by assaying host cultures for loss of the abilityto cleave a chromogenie substrate otherwise hydrolyzed by the hydrolase.

Transformed hosts contained the protease deletion mutants are useful forsynthesis of products which are incompatible with proteolytic enzymes.These hosts by definition are incapable of excreting the deletedproteases described herein, yet are substantially normally sporulating.Also the other growth characteristics of the transformants aresubstantially like the parental organism. Such organisms are useful inthat it is expected they will exhibit comparatively less inactivation ofheterologous proteins than the parents, and these hosts do have growthcharacteristics superior to known protease-deficient organisms. However,the deletion of neutral protease and subtilisin as described in thisapplication does not remove all of the proteolytic activity of Bacillus.It is believed that intracellular proteases which are not ordinarilyexcreted extracellularly "leak" or diffuse from the cells during latephases of the culture. These intracellular proteases may or may not besubtilisin or neutral protease as those enzymes are defined herein.Accordingly, the novel Bacillus strains herein are incapable ofexcreting the subtilisin and/or neutral protease enzymes whichordinarily are excreted extracellularly in the parent strains."Incapable" means not revertible to the wild type. Reversion is a finiteprobability that exists with the heretofore known protease-deficient,naturally occurring strains since there is no assurance that thephenotype of such strains is not a function of a readily revertiblemutation, e.g. a point mutation. This to be contrasted with theextremely large deletions provided herein.

The deletion mutant-transformed host cells herein are free of genesencoding enzymatically active neutral protease or subtilisin, whichgenes are defined as those being substantially homologous with the genesset forth in FIGS. 1, 7 or 10. "Homologous" genes contain coding regionscapable of hybridizing under high stringency conditions with the genesshown in FIGS. 1, 7 or 10.

The microbial strains containing carbonyl hydrolase deletion mutants areuseful in two principal processes. In one embodiment they areadvantageous in the fermentative production of products ordinarilyexpressed by a host that are desirably uncontaminated with the proteinencoded by the deletion gene. An example is fermentative synthesis ofamylase, where contaminant proteases interfere in many industrial usesfor amylase. The novel strains herein relieve the art from part of theburden of purifying such products free of contaminating carbonylhydrolases.

In a-second principal embodiment, subtilisin and neutral proteasedeletion-mutant strains are useful in the synthesis of protein which isnot otherwise encoded by the strain. These proteins will fall within oneof two classes. The first class consists of proteins encoded by genesexhibiting no substantial pretransformation homology with those of thehost. These may be proteins from other procaryotes but ordinarily areeucaryotic proteins from yeast or higher eucaryotic organisms,particularly mammals. The novel strains herein serve as useful hosts forexpressible vectors containing genes encoding such proteins because theprobability for proteolytic degradation of the expressed, non-homologousproteins is reduced.

The second group consists of mutant host genes exhibiting substantialpretransformation homology with those of the host. These includemutations of procaryotic carbonyl hydrolases such as subtilisin andneutral protease, as well as microbial (rennin, for example rennin fromthe genus Mucor). These mutants are selected in order to improve thecharacteristics of the precursor enzyme for industrial uses.

A novel method is provided to facilitate the construction andidentification of such mutants. First, the gene encoding the hydrolaseis obtained and sequenced in whole or in part. Then the sequence isscanned for a point at which it is desired to make a mutation (deletion,insertion or substitution) of one or more amino acids in the expressedenzyme. The sequences flanking this point are evaluated for the presenceof restriction sites for replacing a short segment of the gene with anoligonucleotide pool which when expressed will encode various mutants.Since unique restriction sites are generally not present at locationswithin a convenient distance from the selected point (from 10 to 15nucleotides), such sites are generated by substituting nucleotides inthe gene in such a fashion that neither the reading frame nor the aminoacids encoded are changed in the final construction. The task oflocating suitable flanking regions and evaluating the needed changes toarrive at two unique restriction site sequences is made routine by theredundancy of the genetic code, a restriction enzyme map of the gene andthe large number of different restriction enzymes. Note that if afortuitous flanking unique restriction site is available, the abovemethod need be used only in connection with the flanking region whichdoes not contain a site.

Mutation of the gene in order to change its sequence to conform to thedesired sequence is accomplished by M13 primer extension in accord withgenerally known methods. Once the gene is cloned, it is digested withthe unique restriction enzymes and a plurality of endtermini-complementary oligonucleotide cassettes are ligated into theunique sites. The mutagenesis is enormously simplified by this methodbecause all of the oligonucleotides can be synthesized so as to have thesame restriction sites, and no synthetic linkers are necessary to createthe restriction sites. The number of commercially available restrictionenzymes having sites not present in the gene of interest is generallylarge. A suitable DNA sequence computer search program simplifies thetask of finding potential 5' and 3' unique flanking sites. A primaryconstraint is that any mutation introduced in creation of therestriction site must be silent to the final constructed amino acidcoding sequence. For a candidate restriction site 5' to the target codona sequence must exist in the gene which contains at least all thenucleotides but for one in the recognition sequence 5' to the cut of thecandidate enzyme. For example, the blunt cutting enzyme SmaI (CCC/GGG)would be a 5' candidate if a nearby 5' sequence contained NCC, CNC, orCCN. Furthermore, if N needed to be altered to C this alteration mustleave the amino acid coding sequence intact. In cases where a permanentsilent mutation is necessary to introduce a restriction site one maywant to avoid the introduction of a rarely used codon. A similarsituation for SmaI would apply for 3' flanking sites except the sequenceNGG, GNG, or GGN must exist. The criteria for locating candidate enzymesis most relaxed for blunt cutting enzymes and most stringent for 4 baseoverhang enzymes. In general many candidate sites are available. For thecodon-222 target described herein a Bali site (TGG/CCA) could have beenengineered in one base pair 5' from the KpnI site. A 3' EcoRV site(GAT/ATC) could have been employed 11 base pairs 5' to the PstI site. Acassette having termini ranging from a blunt end up to a fourbase-overhang will function without difficulty. In retrospect, thishypothetical EcoRV site would have significantly shortened theoligonucleotide cassette employed (9 and 13 base pairs) thus allowinggreater purity and lower pool bias problems. Flanking sites shouldobviously be chosen which cannot themselves ligate so that ligation ofthe oligonucleotide cassette can be assured in a single orientation.

The mutation per se need not be predetermined. For example, anoligonucleotide cassette or fragment is randomly mutagenized withnitrosoguanidine or other mutagen and then in turn ligated into thehydrolase gene at a predetermined location.

The mutant carbonyl hydrolases expressed upon transformation of thesuitable hosts are screened for enzymes exhibiting desiredcharacteristics, e.g. substrate specificity, oxidation stability,pH-activity profiles and the like.

A change in substrate specificity is defined as a difference between theKcat/Km ratio of the precursor enzyme and that of the mutant. TheKcat/Km ratio is a measure of catalytic efficiency. Procaryotic carbonylhydrolases with increased or diminished Kcat/Km ratios are described inthe examples. Generally, the objective will be to secure a mutant havinga greater (numerically larger) Kcat/Km ratio for a given substrate,thereby enabling the use of the enzyme to more efficiently act on atarget substrate. An increase in Kcat/Km ratio for one substrate may beis accompanied by a reduction in Kcat/Km ratio for another substrate.This is a shift in substrate specificity, and mutants exhibiting suchshifts have utility where the precursors are undesirable, e.g. toprevent undesired hydrolysis of a particular substrate in an admixtureof substrates.

Kcat and Km are measured in accord with known procedures, or asdescribed in Example 18.

Oxidation stability is a further objective which is accomplished bymutants described in the examples. The stability may be enhanced ordiminished as is desired for various uses. Enhanced stability iseffected by deleting one or more methtonine, tryptophan, cysteine orlysine residues and, optionally, substituting another amino acid residuenot one of methionine, tryptophan, cysteine or lysine. The oppositesubstitutions result in diminished oxidation stability. The substitutedresidue is preferably alanyl, but neutral residues al so are suitable.

Mutants are provided which exhibit modified pH-activity profiles. ApH-activity profile is a plot of pH against enzyme activity and may beconstructed as illustrated in Example 19 or by methods known in the art.It may be desired to obtain mutants with broader profiles, i.e., thosehaving greater activity at certain pH than the precursor, but nosignificantly greater activity at any pH, or mutants with sharperprofiles, i.e. those having enhanced activity when compared to theprecursor at a given pH, and lesser activity elsewhere.

The foregoing mutants preferably are made within the active site of theenzyme as these mutations are most likely to influence activity.However, mutants at other sites important for enzyme stability orconformation are useful. In the case of Bacillus subtilisin or its pre,prepro and pro forms, mutations at tyrosine-1, aspartate+32,asparagine+155, tyrosine+104, methionine+222, glycine+166, histidine+64,glycine+169, phenylalanine+189, serine+33, serine+221, tyrosine+217,glutamate+156 and/or alanine+152 produce mutants having changes in thecharacteristics described above or in the processing of the enzyme. Notethat these amino acid position numbers are those assigned to B.amyloliquefaciens subtilisin as seen from FIG. 7. It should beunderstood that a deletion or insertion in the N-terminal direction froma given position will shift the relative amino acid positions so that aresidue will not occupy its original or wild type numerical position.Also, allelic differences and the variation among various procaryoticspecies will result in positions shifts, so that position 169 in suchsubtilisins will not be occupied by glycine. In such cases the newpositions for glycine will be considered equivalent to and embracedwithin the designation glycine+169. The new position for glycine+169 isreadily identified by scanning the subtilisin in question for a regionhomologous to glycine+169 in FIG. 7.

One or more, ordinarily up to about 10, amino acid residues may bemutated. However, there is no limit to the number of mutations that areto be made aside from commercial practicality.

The enzymes herein may be obtained as salts. It is clear that theionization state of a protein will be dependent on the pH of thesurrounding medium, if it is in solution, or of the solution from whichit is prepared, if it is in solid form. Acidic proteins are commonlyprepared as, for example, the ammonium, sodium, or potassium salts;basic proteins as the chlorides, sulfates, or phosphates. Accordingly,the present application includes both electrically neutral and saltforms of the designated carbonyl hydrolases, and the term carbonylhydrolase refers to the organic structural backbone regardless ofionization state.

The mutants are particularly useful in the food processing and cleaningarts. The carbonyl hydrolases, including mutants, are produced byfermentation as described herein and recovered by suitable techniques.See for example K. Anstrup, 1974, Industrial Aspects of Biochemistry,ed. B. Spencer pp. 23-46. They are formulated with detergents or othersurfactants in accord with methods known per se for use in industrialprocesses, especially laundry. In the latter case the enzymes arecombined with detergents, builders, bleach and/or fluorescent whiteningagents as is known in the art for proteolytic enzymes. Suitabledetergents include linear alkyl benzene sulfonates, alkyl ethoxylatedsulfate, sulfated linear alcohol or ethoxylated linear alcohol. Thecompositions may be formulated in granular or liquid form. See forexample U.S Pat. Nos. 3,623,957; 4,404,128; 4,381,247; 4,404,115;4,318,818; 4,261,868; 4,242,219; 4,142,999; 4,111,855; 4,011,169;4,090,973; 3,985,686; 3,790,482; 3,749,671; 3,560,392; 3,558,498; and3,557,002.

The following disclosure is intended to serve as a representation ofembodiments herein, and should not be construed as limiting the scope ofthis application.

Glossary, of Experimental Manipulations

In order to simplify the Examples certain frequently occurring methodswill be referenced by shorthand phrases.

Plasmids are designated by a small p preceeded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are available on an unrestricted basis, or canbe constructed from such available plasmids in accord with publishedprocedures.

"Klenow treatment" refers to the process of filling a recessed 3' end ofdouble stranded DNA with deoxyribonucleotides complementary to thenucleotides making up the protruding 5' end of the DNA strand. Thisprocess is usually used to fill in a recessed end resulting from arestriction enzyme cleavage of DNA. This creates a blunt or flush end,as may be required for further ligations. Treatment with Klenow isaccomplished by reacting (generally for 15 minutes at 15° C.) theappropriate complementary deoxyribonucleotides with the DNA to be filledin under the catalytic activity (usually 10 units) of the Klenowfragment of E. coli DNA polymerase I ("Klenow"). Klenow and the otherreagents needed are commercially available. The procedure has beenpublished extensively. See for example T. Maniatis et al., 1982,Molecular Cloning, pp. 107-108.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. "Partial" digestion refers to incompletedigestion by a restriction enzyme, i.e., conditions are chosen thatresult in cleavage of some but not all of the sites for a givenrestriction endonuclease in a DNA substrate. The various restrictionenzymes used herein are commercially available and their reactionconditions, cofactors and other requirements as established by theenzyme suppliers were used. Restriction enzymes commonly are designatedby abbreviations composed of a capital letter followed by other lettersand then, generally, a number representing the microorganism from whicheach restriction enzyme originally was obtained. In general, about 1 μgof plasmid or DNA fragment is used with about 1 unit of enzyme in about20 μl of buffer solution. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation times of about 1 hour at 37° C. are ordinarily used, but mayvary in accordance with the supplier's instructions. After incubation,protein is removed by extraction with phenol and chloroform, and thedigested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5' phosphates to prevent the two restriction cleavedends of a DNA fragment from "circularizing" or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5' terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al., Id., pp.133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on 6 percent polyacrylamide gelelectrophoresis, identification of the fragment of interest by molecularweight (using DNA fragments of known molecular weight as markers),removal of the gel section containing the desired fragment, andseparation of the gel from DNA. This procedure is known generally. Forexample, see R. Lawn et al., 1981, "Nucletc Acids Res." 9:6103-6114, andD. Goeddel et al., (1980) "Nucleic Acids Res." 8:4057.

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, Southern analysis shall mean separation of digests on 1 percentagarose and depurination as described by G. Wahl et al., 1979, "Proc.Nat. Acad. Sci. U.S.A." 76:3683-3687, transfer to nitrocellulose by themethod of E. Southern, 1975, "J. Mol. Biol." 98:503-517, andhybridization as described by T. Maniatis et al., 1978, "Cell"15:687-701.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or chromosomalintegrant. Unless otherwise stated, the method used herein fortransformation of E. coli is the CaCl₂ method of Mandel et al., 1970,"J. Mol. Biol." 53:154, and for Bacillus, the method of Anagnostopolouset al., 1961, "J. Bact." 81:791-746.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al., Id., p.146). Unless otherwise stated, ligation was accomplished using knownbuffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5μg of approximately equimolar amounts of the DNA fragments to beligated. Plasmids from the transformants were prepared, analyzed byrestriction mapping and/or sequenced by the method of Messing, et al.,1981, "Nucleic Acids Res." 9:309.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise stated, the alkaline/SDS method ofManiatis et al., Id. p. 90., was used.

"Oligonucleotides" are short length single or double strandedpolydeoxynucleotides which were chemically synthesized by the method ofCrea et al., 1980, "Nucleic Acids Res." 8:2331-2348 (except thatmesitylene nitrotriazole was used as a condensing agent) and thenpurified on polyacrylamide gels.

All literature citations are expressly incorporated by reference.

EXAMPLE 1

Preparation of a Genomic DNA Library from B. amyloliquifaciens andIsolation of its Subtilisin Gene

The known amino acid sequence of the extracellular B. amyloliquefacienspermits the construction of a suitable probe mixture. The sequence ofthe mature subtilisin is included (along with the additional informationcontributed by the present work) in FIG. 1. All codon ambiguity for thesequence of amino acids at position. 117 through 121 is covered by apool of eight oligonucleotides of the sequence ##STR2##

Chromosomal DNA isolated from B. amyloliquefaciens (ATCC No. 23844) asdescribed by J. Marmur, "J. Mol, Biol" 3:208, was partially digested bySau 3A, and the fragments size selected and ligated into the BamH 1 siteof dephosphorylated pBS42. (pBS42 is shuttle vector containing originsof replication effective both in E. coli and Bacillus. It is prepared asdescribed in Example 4.) The Sau3A fragment containing vectors weretransformed into E. col K12 strain 294 (ATCC No. 31446) according to themethod of M. Mandel, et al., 1970, . "J. Mol. Bio." 53:154 using 80-400nanograms of library DNA per 250 μL of competent cells.

Cells from the transformation mixture were plated at a density of1-5×10³ transformants per 150 mm plate containing LB medium+12.5 μg/mlchloramphenicol, and grown overnight at 37° C. until visible coloniesappeared. The plates were then replica plated onto BASS nitrocellulosefilters overlayed on LB/chloramphentcol plates. The replica plates weregrown 10-12 hours at 37° C. and the filters transferred to fresh platescontaining LB and 150 μg/ml spectinomycin to amplify the plasmid pool.

After overnight incubation at 37° C., filters were processed essentiallyas described by Grunstein and Hogness, 1975, "Proc. Natl. Acad. Sci.(USA)" 72: 3961. Out of approximately 20,000 successful transformants,25 positive colonies were found. Eight of these positives were streakedto purify individual clones. 24 clones from each streak were grown inmicrotiter wells, stamped on to two replica filters, and probed asdescribed above with either ##STR3## which differ by only onenucleotide. As shown in FIG. 2, pool 1 hydridized to a much greaterextent to all positive clones than did pool 2, suggesting specifichybridization.

Four out of five miniplasmid preparations (Maniatis et al., Id.) frompositive clones gave identical restriction digest patterns when digestedwith Sau3A or HincII. The plasmid isolated from one of these fouridentical colonies by the method of Maniatis et al., Id., had the entirecorrect gene sequence and was designated pS4. The characteristics ofthis plasmid as determined by restriction analysis are shown in FIG. 3.

EXAMPLE 2 Expression of the Subtilisin Gene

Bacillus subtills I-168 (Catalog No. 1-A1, Bacillus Genetic StockCenter) was transformed with pS4 and and a single chloramphenicolresistant transformant then grown in minimal medium. After 24 hours, theculture was centrifuged and both the supernatant (10-200 μl) and pelletassayed for proteolytic activity by measuring the change in absorbanceper minute at 412 nm using 1 ml of the chromogenic substratesuccinyl-L-ala-ala-pro-phe-p-nitroanilide (0.2 μM) in 0.1M sodiumphosphate (pH 8.0) at 25° C. A B. subtills I-168 culture transformedwith pBS42 used as a control showed less than 1/200 of the activityshown by the pS4 transformed culture. Greater than 95 percent of theprotease activity of the pS4 culture was present in the supernatant, andwas completely inhibited by treatment with phenylmethylsulfonyl fluoride(PMSF) but not by EDTA.

Aliquots of the supernatants were treated with PMSF and EDTA to inhibitall protease activity and analyzed by 12 percent SDS-PAGE according tothe method of Laemmli, U.K., 1970 "Nature" 227: 680. To prepare thesupernatants, 16 L of supernatant was treated with 1mM PMSF, 10 mN EDTAfor 10 minutes, and boiled with 4 μL of 5× concentrated SDS samplebuffer minus β-mercaptoethanol. The results of Coomassie stain on runsusing supernatants of cells transformed with pS4; pBS42, anduntransformed B. amyloliquefaciens are shown in FIG. 4. Lane 3 showsauthentic subtilisin from B. amyloliquefaciens. Lane 2 which is thesupernatant from pBS42 transformed B. subtilis, does not give the 31,000MW band associated with subtilisin which is exhibited by Lane 1 from pS4transformed hosts. The approximately 31,000 MW band result forsubtilisin is characteristic of the slower mobility shown by the knownM.W. 27,500 subtilisin preparations in general.

EXAMPLE 3 Sequencing of the B. amloliquefaciens Subtilisin Gene

The entire sequence of an EcoRI-BamHI fragment (wherein the EcoRI sitewas constructed by conversion of the HincII site) of pS4 was determinedby the method of F. Sanger, 1977, "Proc. Natl. Acad. Sci (USA)" 74:5463.Referring to the restriction map shown in FIG. 3, the BamHI-PvuIIfragment was found to hybridize with pool 1 oligonucleotides by Southernanalysis. Data obtained from sequencing of this fragment directed thesequencing of the remaining fragments (e.g. PvuII-HincII and AvaI-AvaI).The results are shown in FIG. 1.

Examination of the sequence confirms the presence of codons for themature subtilisin corresponding to that secreted by the B.amloliquefaciens. Immediately upstream from this sequence is a series of107 codons beginning with the GTG start codon at -107. Codon -107 toapproximately codon -75 encodes an amino acid sequence whosecharacteristics correspond to that of known signal sequences. (Most suchsignal sequences are 18-30 amino acids in length, have hydrophobiccores, and terminate in a small hydrophobic amino acid.) Accordingly,examination of the sequence data would indicate that codons -107 toapproximately -75 encode the signal sequence; the remaining interveningcodons between -75 and -1 presumably encode a prosequence.

Example 4 Construction of pBS42

pBS42 is formed by three-way ligation of fragments derived from pUB110,pC194, and pBR322 (see FIG. 5). The fragment from pUB110 is theapproximately 2600 base pair fragment between the HpaII site at 1900 andthe BamH1 site at 4500 and contains an origin of replication operable inBacillus: T Grycztan, et al., 1978 "J. Bacteriol" 134: 318 (1978); A.Jalanko, et al., 1981 "Gene", 14: 325. The BamHI site was tested withKlenow. The pBR322 portion is the 1100 base pair fragment between thePvuII site at 2067 and the Sau3A site at 3223 which contains the E. coliorigin of replication: F. Bolivar, et al., 1977 "Gene", 2: 95; J.Sutcliffe 1978 Cold Spring Harbor Symposium 43: I, 77. The pC194fragment is the 1200 base pair fragment between the HpaII site at 973and the Sau3A site at 2006 which contains the gene for chloramphenicolresistance expressible in both E. col and B. subtills: S. Ehrlich, "ProcNatl Acad. Sci (USA)" 74:1680; S. Horynuchi et al., 1982, "J.Bacteriol." 150: 815.

pBS42 thus contains origins of replication operable both in E. coli andin Bacillus and an expressible gene for chloramphenicol resistance.

Example 5 Isolation and Sequencing of the B. subtills Subtilisin Gene

B. subtills I168 chromosomal DNA was digested with EcoRI and thefragments resolved on gel electrophoresis. A single 6 kb fragmenthybridized to a [α-³² P] CTP nick translation--labelled fragmentobtained from the C-terminus of the subtiltsin structural gene in pS4,described above. The 6 kb fragment was electroluted and ligated intopBS42 which had been digested with EcoRI and treated with bacterialalkaline phosphatase. E. coli ATCC 31446 was transformed with theligation mixture and transformants selected by growth on LB agarcontaining 12.5 μg chloramphenicol/ml. Plasmid DNA was prepared from apooled suspension of 5,000 transformed colonies. This DNA wastransformed into B. subtilis BG84, a protease deficient strain, thepreparation of which is described in Example 8 below. Colonies whichproduced protease were screened by plating on LB agar plus 1.5 percentw/w Carnation powdered nonfat skim milk and 5 μg chloramphenicol/ml(hereafter termed skim milk selection plates) and observing for zones ofclearance evidencing proteolytic activity.

Plasmid DNA was prepared from protease producing colonies, digested withEcoRI, and examined by Southern analysis for the presence of the 6 kbEcoRI insert by hybridization to the ³² P-labelled C-terminus fragmentof the subtilisin structural gene from B. amyloliquefaciens. A positiveclone was identified and the plasmid was designated pS168.1. B. subtilisBG84 transformed with pS168.1 excreted serine protease at a level 5-foldover that produced in B. subtills I168. Addition of EDTA to thesupernatants did not affect the assay results, but the addition of PMSF(phenylmethylsufonyl fluoride) to the supernatants reduced proteaseactivity to levels undetectable in the assay described in Example 8 forstrain BG84.

A restriction map of the 6.5 kb EcoRI insert is shown in FIG. 6. Thesubtilisin gene was localized to within the 2.5 kb KpnI-EcoRI fragmentby subcloning various restriction enzyme digests and testing forexpression of subtilisin in B. subtilis BG84. Southern analysis with thelabelled fragment from the C-terminus of the B. amyloliquefacienssubtilisin gene as a probe localized the C-terminus of the B. subtilisgene to within or part of the 631 bp HincII fragment B in the center ofthis subclone (see FIG. 6). The tandem HincII fragments B, C, and D andHincII-EcoRI fragment E (FIG. 6) were ligated into the M13 vectors mp8or mp9 and sequenced in known fashion (J. Messing et al., 1982, "Gene"19:209-276) using dideoxy chain termination (F. Sanger et al., 1977,"Proc. Nat. Acad. Sci. U.S.A." 74:5463-5467). The sequence of thisregion is shown in FIG. 7. The first 23 amino acids are believed to be asignal peptide. The remaining 83 amino acids between the signal sequenceand the mature coding sequence constitute the putative "pro" sequence.The overlined nucleotides at the 3' end of the gene are believed to betranscription terminator regions. Two possible Shine-Dalgarno sequencesare underlined upstream from the mature start codon.

EXAMPLE 6 Manufacture of an Inactivating Mutation of the B. subtilisSubtilisin Gene

A two step ligation, shown in FIG. 8, was required to construct aplasmid carrying a defective gene which would integrate into theBacillus chromosome. In the first step, pS168.1, which contained the 6.5kb insert originally recovered from the B. subtilis genomic library asdescribed in Example 5 above, was digested with EcoRI, the reactionproducts treated with Klenow, the DNA digested with HincII, and the 800bp EcoRI-HincII fragment E (see FIG. 6) that contains, in part, the 5'end of the B. subtilis subtilisin gene, was recovered. This fragment wasligated into pJH101 (pjH101 is available from J. Hoch (Scripps) and isdescribed by F. A. Ferrari et al., 1983, "J. Bact." 134:318-329) thathad been digested with HincII and treated with bacterial alkalinephosphotase. The resultant plasmid, pIDV1, contained fragment E in theorientation shown in FIG. 8. In the second step, pS168.1 was digestedwith HincII and the 700 bp HincII fragment B, which contains the 3' endof the subtilisin gene, was recovered. pIDV1 was digested at its uniqueHincII site and fragment B ligated to the linearized plasmid,transformed in E. coli ATCC 31,446, and selected on LB plates containing12.5 μg chloramphenicol/ml or 20 μg ampicillin/ml. One resultingplasmid, designated pIDV1.4, contained fragment B in the correctorientation with respect to fragment E. This plasmid pIDV1.4, shown inFIG. 8, is a deletion derivative of the subtilisin gene containingportions of the 5' and 3' flanking sequences as well.

B. subtilis BG77, a partial protease-deficient mutant (Prt.sup.±)prepared in Example 8 below was transformed with pIDV1.4. Two classes ofchloramphenicol resistant (Cm^(r)) transformants were obtained.Seventy-five percent showed the same level of proteases as BG77(Prt.sup.±) and 25 percent were almost completely protease deficient(Prt⁻) as observed by relative zones of clearing on plates containing LBagar plus skim milk. The Cm^(r) Prt⁻ transformants could not be due to asingle crossover integration of the plasmid at the homologous regionsfor fragment E or B because, in such a case, the gene would beuninterrupted and the phenotype would be Prt.sup.±. In fact, when eitherof fragments E or B were ligated independently into pJH101 andsubsequently transformed into B. subtilis BG77, the protease deficientphenotype was not observed. The Cm^(r) phenotype of Cm^(r) Prt⁻ pIDV1.4transformants was unstable in that Cm^(s) Prt⁻ derivatives could beisolated from Cm^(r) Prt⁻ cultures at a frequency of about 0.1 percentafter 10 generations of growth in minimal medium in the absence ofantibiotic selection. One such derivative was obtained and designatedBG2018. The deletion was transferred into IA84 (a BGSC strain carryingtwo auxotrophic mutations flanking the subtilisin gene) by PBS1transduction. The derivative organism was designated BG2019.

EXAMPLE 7 Preparation of a Genomic DNA Library from B. subtilis andIsolation of its Neutral Protease Gene

The partial amino acid sequence of a neutral protease of B. subtilis isdisclosed by P. Levy et al. 1975, "Proc. Nat. Acad. Sci. USA"72:4341-4345. A region of the enzyme (Asp Gln Met Ile Tyr Gly) wasselected from this published sequence in which the least redundancyexisted in the potential codons for the amino acids in the region. 24combinations were necessary to cover all the potential coding sequences,as described below. ##STR4##

Four pools, each containing six alternatives, were prepared as describedabove in Example 1. The pools were labelled by phosphorylization with[γ³² p] ATP.

The labelled pool containing sequences conforming closest to a uniquesequence in a B. subtilis genome was selected by digesting B. subtilis(1A72, Bacillus Genetic Stock Center) DNA with various restrictionenzymes, separating the digests on an electrophoresis gel, andhybridizing each of the four probe pools to each of the blotted digestsunder increasingly stringent conditions until a single band was seen tohybridize. Increasingly stringent conditions are those which tend todisfavor hybridization, e.g., increases in formamide concentration,decreases in salt concentration and increases in temperature. At 37° C.in a solution of 5× Denhardt's, 5× SSC, 50 mM NaPO₄ pH 6.8 and 20percent formamide, only pool 4 would hybridize to a blotted digest.These were selected as the proper hybridization conditions to be usedfor the neutral protease gene and pool 4 was used as the probe.

A lambda library of B. subtilis strain BGSC 1-A72 was prepared inconventional fashion by partial digestion of the Bacillus genomic DNA bySau3A, separation of the partial digest by molecular weight on anelectrophoresis gel, elution of 15-20 kb fragments (R. Lawn et al.,1981, "Nucleic Acids Res." 9:6103-6114), and ligation of the fragmentsto BamHI digested charon 30 phage using a Packagene kit from PromegaBiotec.

E. coli DP50supF was used as the host for the phage library, althoughany known host for Charon lambda phage is satisfactory. The E. coli hostwas plated with the library phage and cultured, after which plaques wereassayed for the presence of the neutral protease gene by transfer tonitrocellulose and screening with probe pool 4 (Benton and Davis, 1977,"Science" 196:180-182). Positive plaques were purified through tworounds of single plaque purification, and two plaques were chosen forfurther study, designated λNPRG1 and λNPRG2. DNA was prepared from eachphage by restriction enzyme hydrolysis and separation on electrophoresisgels. The separated fragments were blotted and hybridized to labelledpool 4 oligonucleotides. This disclosed that λNPRG1 contained a 2400 bpHindIII hybridizing fragment, but no 4300 EcoRI fragment, while λNPRG2contained a 4300 bp EcoRI fragment, but no 2400 bp HindIII fragment.

The 2400 bp λNPRG1 fragment was subcloned into the HindIII site ofpJH101 by the following method. λNPRG1 was digested by HindIII, thedigest fractionated by electrophoresis and the 2400 bp fragmentrecovered from the gel. The fragment was ligated to alkalinephospate-treated Hind-III digested pJH101 and the ligation mixture usedto transform E. coli ATCC 31446 by the calcium chloride shock recoveredfrom the gel. The fragment was ligated to alkalin method of V.Hershfield et al., 1974, "Proc. Nat. Acad. Sci. (U.S.A.)" 79:3455-3459).Transformants were identified by selecting colonies capable of growth onplates containing LB medium plus 12.5 μg chloramphenicol/ml.

Transformant colonies yielded several plasmids. The orientation of the2400 bp fragment in each plasmid was determined by conventionalrestriction analysis (orientation is the sense reading ortranscriptional direction of the gene fragment in relation to thereading direction of the expression vector into which it is ligated.)Two plasmids with opposite orientations were obtained and designatedpNPRsubH6 and pNPRsubH1.

The 4300 bp EcoRI fragment of λNPRG2 was subcloned into pBR325 by themethod described above for the 2400 bp fragment except that λNPRG2 wasdigested with EcoRI and the plasmid was alkaline phosphatase-treated,EcoRI-digested pBR325. pBR325 is described by F. Bolivar, 1978, "Gene"4:121-136. Two plasmids were identified in which the 4300 bp insert waspresent in different orientations. These two plasmids were designatedpNPRsubRI and pPRsubRIb.

EXAMPLE 8 Characterization of B. subtilis Neutral Protease Gene

The pNPRsubH1 insert was sequentially digested with differentrestriction endonucleases and blot hybridized with labelled pool 4 inorder to prepare a restriction map of the insert (for general proceduresof restriction mapping see T. Maniatis et al., Id., p. 377). A 430 bpRsaI fragment was the smallest fragment that hybridized to probe pool 4.The RsaI fragment was ligated into the SmaI site of M13 mp8 (J. Messinget al., 1982, "Gene" 19:269-276 and J. Messing in Methods in Enzymology,1983, R. Wu et al., Eds., 101:20-78) and the sequence determined by thechain-terminating dideoxy method (F. Sanger et al., 1977, "Proc. Nat.Acad. Sci. U.S.A." 74:5463-5467). Other restriction fragments from thepNPRsubH1 insert were ligated into appropriate sites in M13 mp8 or M13mp9 vectors and the sequences determined. As required, dITP was used toreduce compression artifacts (D. Mills et al., 1979, "Proc. Nat. Acad.Sci. (U.S.A.)" 76:2232-2235). The restriction map for the pNPRsubH1fragment is shown in FIG. 9. The sequences of the various fragments fromrestriction enzyme digests were compared and an open reading framespanning a codon sequence translatable into the amino and carboxyltermini of the protease (P. Levy et al., Id. ) was determined. An openreading frame is a DNA sequence commencing at a known point which inreading frame (every three nucleotides) does not contain any internaltermination codons. The open reading frame extended past the aminoterminus to the end of the 2400 bp HindIII fragment. The 1300 bp BglII -HindIII fragment was prepared from pNPRsubRIb (which contained the 4300bp EcoRI fragment of λNPRG2) and cloned in M13 mp8. The sequence of thisfragment, which contained the portion of the neutral protease leaderregion not encoded by the 2400 bp fragment of pNPRsubH1, was determinedfor 400 nucleotides upstream from the HindIII site.

The entire nucleotide sequence as determined for this neutral proteasegene, including the putative secretory leader and prepro sequence, areshown in FIG. 10. The numbers above the line refer to amino acidpositions. The underlined nucleotides in FIG. 10 are believed toconstitute the ribosome binding (Shine-Dalgarno) site, while theoverlined nucleotides constitute a potential hairpin structure presumedto be a terminator. The first 27-28 of the deduced amino acids arebelieved to be the signal for the neutral protease, with a cleavagepoint at ala-27 or ala-28. The "pro" sequence of a proenzyme structureextends to the amino-terminal amino acid (ala-222) of the mature, activeenzyme.

A high copy plasmid carrying the entire neutral protease gene wasconstructed by (FIG. 11) ligating the BglII fragment of pNPRsubR1, whichcontains 1900 bp (FIG. 9), with the PvuII - HindIII fragment ofpNPRsubH1, which contains 1400 bp. pBS42 (from Example 4) was digestedwith BamHI and treated with bacterial alkaline phosphatase to preventplasmid recircularization. pNPRsubR1 was digested with BglII, the 1900bp fragment was isolated from gel electrophoresis and ligated to theopen BamHI sites of pBS42. The ligated plasmid was used to transform E.coli ATCC 31446 by the calcium chloride shock method (V. Hershfield etal., Id.), and transformed cells selected by growth on plates containingLB medium with 12.5 μg/ml chloramphenicol. A plasmid having the BglIIfragment in the orientation shown in FIG. 11 was isolated from thetransformants and designated pNPRsubB1. pNPRsubB1 was digested(linearized) with EcoRI, repaired to flush ends by Klenow treatment andthen digested with HindIII. The larger fragment from the HindIIIdigestion (containing the sequence coding for the amino terminal andupstream regions) was recovered.

The carboxyl terminal region of the gene was supplied by a fragment frompNPRsubH1, obtained by digestion of pNPRsubH1 with PvuII and HindIII andrecovery of the 1400 bp fragment. The flush end PvuII and the HindIIIsite of the 1400 bp fragment was ligated, respectively, to the bluntedEcoRI and the HindIII site of pNPRsubB1, as shown in FIG. 11. Thisconstruct was used to transform B. subtilis strain BG84 which otherwiseexcreted no proteolytic activity by the assays described below.Transformants were selected on plates containing LB medium plus 1.5percent carnation powdered nonfat milk and 5 μg/ml chloramphenicol.Plasmids from colonies that cleared a large halo were analyzed. PlasmidpNPR10, incorporating the structural gene and flanking regions of theneutral protease gene, was determined by restriction analysis to havethe structure shown in FIG. 11.

B. subtills strain BG84 was produced byN-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis of B subtillsI168 according to the general technique of Adelberg et al., 1965,"Biochem. Biophys. Res. Commun." 18:788-795. Mutagenized strain I168 wasplated on skim milk plates (without antibiotic). Colonies producing asmaller halo were picked for further analysis. Each colony wascharacterized for protease production on skim milk plates and amylaseproduction on starch plates. One such isolate, which was partiallyprotease deficient, amylase positive and capable of sporulation, wasdesignated BG77. The protease deficiency mutation was designated prt-77.The prt-77 allele was moved to a spo0A background by congression asdescribed below to produce strain BG84, a sporulation deficient strain.

                  TABLE A                                                         ______________________________________                                        Strain                                                                              Relevant Genotype       origin                                          ______________________________________                                        I168                                                                                 ##STR5##                                                               JH703                                                                                ##STR6##               Trousdale et al..sup.a                          BG16                                                                                 ##STR7##               Pb 1665                                               sacA321                                                                 BG77                                                                                 ##STR8##               NTG × I168                                BG81                                                                                 ##STR9##               BG16 DNA × BG77                           BG84                                                                                 ##STR10##              JH703 DNA × BG81                          ______________________________________                                         ##STR11##                                                                

BG84 was completely devoid of protease activity on skim milk plates anddoes not produce detectable levels of either subtilisin or neutralprotease when assayed by measuring the change in absorbance at 412 nmper minute upon incubation with 0.2 μg/ml succinyl(-L-ala-L-ala-L-pro-L-phe) p-nitroanilide (Vega) in 0.1M sodiumphosphate, pH 8, at 25° C. BG84 was deposited in the ATCC as depositnumber 39382 on Jul. 21, 1983. Samples for subtilisin assay were takenfrom late logarithmic growth phase supernatants of cultures grown inmodified Schaeffer's medium (T. Leighton et al., 1971, "J. Biol. Chem."246:3189-3195).

EXAMPLE 9 Expression of the Neutral Protease Gene.

BG84 transformed with pNPR10 was inoculated into minimal mediasupplemented with 0.1 percent casein hydrolysate and 10 μgchloramphenicol and cultured for 16 hours. 0.1 ml of culture supernatantwas removed and added to a suspension of 1.4 mg/ml Azocoll proteolyticsubstrate (Sigma) in 10 mM Tris-HCl, 100 mM NaCl pH 6.8 and incubatingwith agitation. Undigested substrate was removed by centrifugation andthe optical density read at 505 nm. Background values of an Azocollsubstrate suspension were subtracted. The amount of protease excreted bya standard protease-expressing strain, BG16 was used to establish anarbitrary level of 100. The results with BG16, and with BG84 transformedwith control and neutral protease gene-containing plasmids are shown inTable B in Example 12 below. Transformation of the excretedprotease-devoid B. subtilis strain BG84 results in excretion of proteaseactivity at considerably greater levels than in BG16, the wild-typestrain.

EXAMPLE 10 Manufacture of an Inactivating Mutation of the NeutralProtease Gene

The two RsaI bounded regions in the 2400 bp insert of pNPRsubH1,totalling 527 bp, can be deleted in order to produce an incompletestructural gene. The translational products of this gene areenzymatically inactive, A plasmid having this deletion was constructedas follows, pJH101 was cleaved by digestion with HindIII and treatedwith bacterial alkaline phosphatase. The fragments of the neutralprotease gene to be incorporated into linearized pJH101 were obtained bydigesting pNPRsubH1 with HindIII and RsaI, and recovering the 1200 bpHindIII-RsaI and 680 bp RsaI-HindIII fragments by gel electrophoresis.These fragments were ligated into linearized pJH101 and used totransform E. coli ATCC 31446. Transformants were selected on platescontaining LB medium and 20 μg ampicillin/ml. Plasmids were recoveredfrom the transformants and assayed by restriction enzyme analysis toidentify a plasmid having the two fragments in the same orientation asin the pNPRsubH1 starting plasmid. The plasmid lacking the internal RsaIfragments was designated pNPRsubH1Δ.

EXAMPLE 11 Replacement of the Neutral Protease Gene with a DeletionMutant

Plasmid pNPRsubh1Δ was transformed into B. subtilis strain BG2019 (thesubtilisin deleted mutant from Example 6) and chromosomal integrantswere selected on skim milk plates. Two types of Cm^(r) transformantswere noted, those with parental levels of proteolysis surrounding thecolony, and those with almost no zone of proteolysis. Those lacking azone of proteolysis were picked, restreaked to purify individualcolonies, and their protease deficient character on skim milk platesconfirmed. One of the Cm^(r), proteolysis deficient colonies was chosenfor further studies (designated BG2034). Spontaneous Cm^(s) revertantsof BG2034 were isolated by overnight growth in LB media containing noCm, plating for individual colonies, and replica plating on media withand without Cm. Three Cm^(s) revertants were isolated, two of which wereprotease proficient, one of which was protease deficient (designatedBG2036). Hybridization analysis of BG2036 confirmed that the plasmid hadbeen lost from this strain, probably by recombination, leaving only thedeletion fragments of subtilisin and neutral protease.

EXAMPLE 12 Phenotype of Strains Lacking Functional Subtilisin andNeutral Protease

The growth, sporulation and expression of proteases was examined instrains lacking a functional gene for either the neutral or alkalineprotease or both. The expression of proteases was examined by a zone ofclearing surrounding a colony on a skim milk plate and by measurement ofthe protease levels in liquid culture supernatants (Table B). A strain(BG2035) carrying the subtilisin gene deletion, and showed a 30 percentreduction level of protease activity and a normal halo on milk plates.Strain BG2043, carrying the deleted neutral protease gene and activesubtilisin gene, and constructed by transforming BG16 (Ex. 8) with DNAfrom BG2036 (Example 11), showed an 80 percent reduction in proteaseactivity and only a small halo on the milk plate. Strain BG2054,considered equivalent to BG2036

                  TABLE B                                                         ______________________________________                                        Effect of protease deletions on protease                                      expression and sporulation.                                                                        Protease Percent                                                 Genotype.sup.a                                                                             activity.sup.b                                                                         Sporulation                                     ______________________________________                                        BG16      Wild type      100      40                                          BG2035    aprΔ684  70       20                                          BG2043    nprEΔ522 20       20                                          BG2054    aprΔ684,nprEΔ522                                                                 ND       45                                          BG84(pBS42)                                                                             spoOAΔ677,prt-77                                                                       ND       --                                          BG84(pNPR10)                                                                            spoOAΔ677,prt-77                                                                       3000     --                                          ______________________________________                                         .sup.a Only the loci relevant to the protease phenotype are shown.            .sup.b Protease activity is espressed in arbitrary units, BG16 was            assigned a level of 100. ND indicates the level of protease was not           detectable in the assay used.                                            

(Example 11) in that it carried the foregoing deletions in both genes,showed no detectable protease activity in this assay and no detectablehalo on milk plates. The deletion of either or both of the proteasegenes had no apparent effect on either growth or sporulation. Strainscarrying these deletions had normal growth rates on both minimal glucoseand LB media. The strains sporulated at frequencies comparable to theparent strain BG16. Examination of morphology of these strains showed noapparent differences from strains without such deletions.

EXAMPLE 13 Site-specific Saturation Mutagenesis of the B.Amyloliquefaciens Subtilisin Gene at Position 222; Preparation of theGene for Cassette Insertion

pS4-5, a derivative of pS4 made according to Wells et al., "NucleicAcids Res" 1983 11:7911-7924 was digested with EcoRI and BamHI, and the1.5 kb EcoRI-BamHI fragment recovered. This fragment was ligated intoreplicative form M-13 mp9 which had been digested with EcoRI and BamHI(Sanger et al., 1980, "J. Mol. Biol. " 143 161-178. Messing et al, 1981,"Nucleic Acids Research" 9, 304-321. Messing, J. and Vieira, J. (1982)Gene 19, 269-276). The M-13 mp9 phage ligations, designated M-13 mp9SUBT, were used to transform E. coli strain JM101 and single strandedphage DNA was prepared from a two mL overnight culture. Anoligonucleotide primer was synthesized having the sequence5'-GTACAACGGTACCTCACGCACGCTGCAGGAGCGGCTGC-3'. This primer conforms tothe sequence of the subtilis gene fragment encoding amino acids 216-232except that the 10 bp of codons for amino acids 222-225 were deleted,and the codons for amino acids 220, 227 and 228 were mutated tointroduce a KpnI site 5' to the met-222 codon and a PstI site 3' to themet+222 codon. See FIG. 12. Substituted nucleotides are denoted byasterisks, the underlined codons in line 2 represent the new restrictionsites and the scored sequence in line 4 represents the insertedoligonucleotides. The primer (about 15 μm) was labelled with [³² p] byincubation with [γ³² p]-ATP (10 μL in 20 μL reaction) (Amersham 5000Ci/mmol, 10218) and T₄ polynucleotide kinase (10 units) followed bynon-radioactive ATP (100 μM) to allow complete phosphorylation of themutagenesis primer. The kinase was inactivated by heating thephosphorylation mixture at 68° C. for 15 min.

The primer was hybridized to M-13 mp9 SUBT as modified from Norris etal., 1983, "Nucleic Acids Res." 11, 5103-5112 by combining 5 μL of thelabelled mutagenesis primer (.sup.˜ 3 M), .sup.˜ 1 μg M-13 mp9 SUBTtemplate, 1 μL of 1 μM M-13 sequencing primer (17-mer), and 2.5 μL ofbuffer (0.3 M Tris pH 8, 40 mM MgCl₂, 12 mM EDTA, 10 mM DTT, 0.5 mg/mlBSA). The mixture was heated to 68° C. for 10 minutes and cooled 10minutes at room temperature. To the annealing mixture was added 3.6 μLof 0.25 mM dGTP, dCTP, dATP, and dTTP, 1.25 μL of 10 mM ATP, 1 μL ligase(4 units) and 1 μL Klenow (5 units). The primer extension and ligationreaction (total volume 25, μl) proceeded 2 hours at 14° C. The Klenowand ligase were inactivated by heating to 68° C. for 20 min. The heatedreaction mixture was digested with BamH1 and EcoRI and an aliquot of thedigest was applied to a 6 percent polyacrylamide gel and radioactivefragments were visualized by autoradiography. This showed the [³² P]mutagenesis primer had indeed been incorporated into the EcoRI-BamH1fragment containing the now mutated subtilisin gene.

The remainder of the digested reaction mixture was diluted to 200 μLwith 10 mM Tris, pH 8, containing 1 mM EDTA, extracted once with a 1:1(v:v) phenol/chloroform mixture, then once with chloroform, and theaqueous phase recovered. 15 μL of 5M ammonium acetate (pH 8) was addedalong with two volumes of ethanol to precipitate the DNA from theaqueous phase. The DNA was pelleted by centrifugation for five minutesin a microfuge and the supernatant was discarded. 300 μL of 70 percentethanol was added to wash the DNA pellet, the wash was discarded and thepellet lyophilized.

pBS42 from example 4 above was digested with BamH1 and EcoRI andpurified on an acrylamide gel to recover the vector. 0.5 μg of thedigested vector, 50 μM ATP and 6 units ligase were dissolved in 20 μl ofligation buffer. The ligation went overnight at 14° C. The DNA wastransformed into E. coli 294 rec⁺ and the transformants grown in 4 ml ofLB medium containing 12.5 μg/ml chloramphenicol. Plasmid DNA wasprepared from this culture and digested with KpnI, EcoRI and BamHI.Analysis of the restriction fragments showed 30-50 percent of themolecules contained the expected KpnI site programmed by the mutagenesisprimer. It was hypothesized that the plasmid population not includingthe KpnI site resulted from M-13 replication before bacterial repair ofthe mutagenesis site, thus producing a heterogenous population of KpnI⁺and KpnI⁻ plasmids in some of the transformants. In order to obtain apure culture of the KpnI⁺ plasmid, the DNA was transformed a second timeinto E. coli to clone plasmids containing the new KpnI site. DNA wasprepared from 16 such transformants and six were found to contain theexpected KpnI site.

Preparative amounts of DNA were made from one of these six transformants(designated pΔ222) and restriction analysis confirmed the presence andlocation of the expected KpnI and PstI sites. 40 μg of pΔ222 weredigested in 300 μL of KpnI buffer plus 30 μL KpnI (300 units) for 1.5 hat 37° C. The DNA was precipitated with ethanol, washed with 70 percentethanol, and lyophilized. The DNA pellet was taken up in 200 μL HindIIIbuffer and digested with 20 μL (500 units) PstI for 1.5 h at 37° C. Theaqueous phase was extracted with phenol/CHCl₃ and the DNA precipitatedwith ethanol. The DNA was dissolved in water and purified bypolyacrylamide gel electrophoresis. Following electroelution of thevector band (120 v for 2 h at 0° C. in 0.1 times TBE (Maniatis et al.,Id.)) the DNA was purified by phenol/CHCl₃ extraction, ethanolprecipitation and ethanol washing.

Although pΔ222 could be digested to completion (>98 percent) by eitherKnpI or PstI separately, exhaustive double digestion was incomplete(<<50 percent). This may have resulted from the fact that these siteswere so close (10 bp) that digestion by KnpI allowed "breathing" of theDNA in the vicinity of the PstI site, i.e., strand separation orfraying. Since PstI will only cleave double stranded DNA, strandseparation could inhibit subsequent PstI digestion.

EXAMPLE 14 Ligation of Oligonucleotide Casettes into the Subtilisin Gene

10 μM of four complementary oligonucleotide pools (A-D, Table 1 below)which were not 5' phosphorylated were annealed in 20 μl ligase buffer byheating for five minutes at 68° C. and then cooling for fifteen minutesat room temperature. 1 μM of each annealed oligonucleotide pool, .sup.˜0.2 μg KpnI and PstI-digested pΔ222 obtained in Example 13, 0.5 mM ATP,ligase buffer and 6 units T₄ DNA ligase in 20 μL total volume wasreacted overnight at 14° C. to ligate the pooled cassettes in thevector. A large excess of cassettes (.sup.˜ 300× over the pΔ222 ends)was used in the ligation to help prevent intramolecular KpnI-KpnIligation. The reaction was diluted by adding 25 μL of 10 mM Tris pH 8containing 1 mM EDTA. The mixture was reannealed to avoid possiblecassette concatemer formation by heating to 68° C. for five minutes andcooling for 15 minutes at room temperature. The ligation mixtures fromeach pool were transformed separately into E. coli 294 rec⁺ cells. Asmall aliquot from each transformation mixture was plated to determinethe number of independent transformants. The large number oftransformants indicated a high probability of multiple mutagenesis. Therest of the transformants (.sup.˜ 200-400 transformants) were culturedin 4 ml of LB medium plus 12.5 μg chloramphenicol/ml. DNA was preparedfrom each transformation pool (A-D). This DNA was digested with KpnI,.sup.˜ 0.1 μg was used to retransform E. coli rec⁺ and the mixture wasplated to isolate individual colonies from each pool. Ligation of thecassettes into the gene and bacterial repair upon transformationdestroyed the KpnI and PstI sites. Thus, only pΔ222 was cut when thetransformant DNA was digested with KpnI. The cut plasmid would nottransform E. coli. Individual transformants were grown in culture andDNA was prepared from 24 to 26 transformants per pool for direct plasmidsequencing. A synthetic oligonucleotide primer having the sequence5'-GAGCTTGATGTCATGGC-3' was used to prime the dideoxy sequencingreaction. The mutants which were obtained are described in Table Cbelow.

Two codon+222 mutants (i.e., gln and ile) were not found after thescreening described. To obtain these a single 25 mer oligonucleotide wassynthesized for each mutant corresponding to the top oligonucleotidestrand in FIG. 12. Each was phosphorylated and annealed to the bottomstrand of its respective nonphosphorylated oligonucleotide pool (i.e.,pool A for gln and pool D for ile). This was ligated into KpnI and PstIdigested pΔ222 and processed as described for the originaloligonucleotide pools. The frequency of appearance for single mutantsobtained this way was 2/8 and 0/7 for gln and ile, respectively. Toavoid this apparent bias the top strand was phosphorylated and annealedto its unphosphorylated complementary pool. The heterophosphorylatedcassette was ligated into cut pΔ222 and processed as before. Thefrequency of appearance of gln and ile mutants was now 7/7 and 7/7,respectively.

The data in Table C demonstrate a bias in the frequency of mutantsobtained from the pools. This probably resulted from unequalrepresentation of oligonucleotides in the pool. This may have beencaused by unequal coupling of the particular trimers over themutagenesis codon in the pool. Such a bias problem could be remedied byappropriate adjustment of trimer levels during synthesis to reflectequal reaction. In any case, mutants which were not isolated in theprimary screen were obtained by synthesizing a single strandoligonucleotide representing the desired mutation, phosphorylating bothends, annealing to the pool of non-phosphorylated complementary strandsand ligating into the cassette site. A biased heteroduplex repairobserved for the completely unphosphorylated cassette may result fromthe fact that position 222 is closer to the 5' end of the upper strandthan it is to the 5' end of the lower strand (see FIG. 12). Because agap exists at the unphosphorylated 5' ends and the mismatch bubble inthe double stranded DNA is at position 222, excision repair of the topstrand gap would more readily maintain a circularly hybridized duplexcapable of replication. Consistent with this hypothesis is the fact thatthe top strand could be completely retained by selective 5'phosphorylation. In this case only the bottom strand contained a 5' gapwhich could promote excision repair. This method is useful in directingbiased incorporation of synthetic oligonuclotide strands when employingmutagenic oligonucleotide cassettes.

EXAMPLE 15 Site-Specific Mutagenesis of the Subtilisin Gene at Position166

The procedure of Examples 13-14 was followed in substantial detail,except that the mutagenesis primer differed (the 37 mer shown in FIG. 13was used), the two restriction enzymes were SacI and XmaIII rather thanPstI and KpnI and the resulting constructions differed, as shown in FIG.13.

Bacillus strains excreting mutant subtilisins at position 166 wereobtained as described below in Example 16. The mutant subtilisinsexhibiting substitutions of ala, asp, gln, phe, his, lys, asn, arg, andval for the wild-type residue were recovered.

EXAMPLE 16 Preparation of Mutant Subtilisin Enzymes

B. subtilis strain BG2036 obtained by the method of Example 11 wastransformed by the plasmids of Examples 14, 15 or 20 and by pS4-5 as acontrol. Transformants were plated or cultured in shaker flasks for 16to 48 h at 37° C. in LB media plus 12.5 μg/ml chloramphenicol. Mutantenzymatically active subtilisin was recovered by dialyzing cell brothagainst 0.01M sodium phosphate buffer, pH 6.2. The dialyzed broth wasthen titrated to pH 6.2 with 1N HCl and loaded on a 2.5×2 cm column ofCM cellulose (CM-52 Whatman). After washing with 0.01M sodium phosphate,pH 6.2, the subtilisins (except mutants at position +222) were elutedwith the same buffer made 0.08N in NaCl. The mutant subtilisins atposition +222 were each eluted with 0.1M sodium phosphate, pH 7.0. Thepurified mutant and wild type enzymes were then used in studies ofoxidation stability, Km, Kcat, Kcat/Km ratio, pH optimum, and changes insubstrate specificity.

                  TABLE C                                                         ______________________________________                                        Oligonucleotide Pool Organization                                             and Frequency of Mutants Obtained                                             Pool   Amino Acids     Codon-222.sup.a                                                                         Frequency.sup.b                              ______________________________________                                        A      asp             GAT       2/25                                                met             ATG       3/25                                                cys             TGT       13/25                                               arg             AGA       2/25                                                gln             GAA       0/25                                                unexpected mutants.sup.a  5/25                                         B      leu             CTT       1/25                                                pro             CCT       3/25                                                phe             TTC       6/25                                                tyr             TAC       5/25                                                his             CAC       1/25                                                unpexpected mutants       9/25                                         C      glu             GAA       3/17                                                ala             GCT       3/17                                                thr             ACA       1/17                                                lys             AAA       1/17                                                asn             AAC       1/17                                                unexpected mutants        8/17                                         D      gly             GGC       1/23                                                trp             TGG       8/23                                                ile             ATC       0/23                                                ser             AGC       1/23                                                val             GTT       4/23                                                unexpected mutants        9/23                                         ______________________________________                                         .sup.a Condons were chosen on frequent use in the cloned subtilisin gene      sequence (Wells et al., 1983, Id.).                                           .sup.b Frequency was determined from single track analysis by direct          plasmid sequencing.                                                           .sup.c Unexpected mutants generally comprised double mutants with changes     in codons next to 222 or at the points of ligation. These were believed t     result from impurities in the obigonucleotide pools and/or erroneous          repair of the gapped ends.                                               

EXAMPLE 17 Mutant Subtilisin Exhibiting Improved Oxidation Stability

Subtilisins having cysteine and alanine substituted at the 222 positionfor wild-type methionine (Example 16) were assayed for resistance tooxidation by incubating with various concentrations of sodiumhypochloride (Clorox Bleach).

To a total volume of 400 μl of 0.1M, pH 7, NaPO₄ buffer containing theindicated bleach concentrations (FIG. 14) sufficient enzyme was added togive a final concentration of 0.016 mg/ml of enzyme. The solutions wereincubated at 25° C. for 10 min. and assayed for enzyme activity asfollows: 120 μl of either ala+222 or wild type, or 100 μl of the cys+222incubation mixture was combined with 890 μl 0.1M tris buffer at pH 8.6and 10 μl of a sAAPFpN (Example 18) substrate solution (20 mg/ml inDMSO). The rate of increase in absorbance at 410 nm due to release ofp-nitroaniline (Del Mar, E. G., et al., 1979 "Anal. Biochem." 99,316-320) was monitored. The results are shown in FIG. 14. The alaninesubstitution produced considerably more stable enzyme than either thewild-type enzyme or a mutant in which a labile cysteine residue wassubstituted for methionine. Surprisingly, the alanine substitution didnot substantially interfere with enzyme activity against the assaysubstrate, yet conferred relative oxidation stability on the enzyme. Theserine+222 mutant also exhibited improved oxidation stability.

EXAMPLE 18 Mutant Subtilisins Exhibiting Modified Kinetics and SubstrateSpecificity

Various mutants for glycine+166 were screened for modified Kcat, Km andKcat/Km ratios. Kinetic parameters were obtained by analysis of theprogress curves of the reactions. The rate of rection was measured as afunction of substrate concentration. Data was analyzed by fitting to theMichaelis-Menton equation using the non-linear regression algorithm ofMarquardt (Marquardt, D. W. 1963, "J. Soc. Ind. Appl. Math." 11,431-41). All reactions were conducted at 25° C. in 0.1M tris buffer, pH8.6, containing benzoyl-L-Valyl-Glycyl-L-Arginyl-p-nitroanilide (BVGRpN;Vega Biochemicals) at initial concentrations of 0.0025M to 0.00026M(depending on the value of Km for the enzyme of interest--concentrationswere adjusted in each measurement so as to exceed Km) orsuccinyl-L-Alanyl-L-Alanyl-L-Prolyl-L-Phenylalanyl-p-nitroanilide(sAAPFpN; Vega Biochemicals) at initial concentrations of 0.0010M to0.00028M (varying as described for BVGRpN).

The results obtained in these experiments were as follows:

                  TABLE D                                                         ______________________________________                                                             Kcat                                                     Substrate                                                                             Enzyme       (s.sup.-1)                                                                             Km(M)   Kcat/Km                                 ______________________________________                                        sAAPFpN gly-166(wild type)                                                                         37       1.4 × 10.sup.-4                                                                 3 × 10.sup.5                              ala + 166    19       2.7 × 10.sup.-5                                                                 7 × 10.sup.5                              asp + 166    3        5.8 × 10.sup.-4                                                                 5 × 10.sup.3                              glu + 166    11       3.4 × 10.sup.-4                                                                 3 × 10.sup.4                              phe + 166    3        1.4 × 10.sup.-5                                                                 2 × 10.sup.5                              hys + 166    15       1.1 × 10.sup.-4                                                                 1 × 10.sup.5                              lys + 166    15       3.4 × 10.sup.-5                                                                 4 × 10.sup.5                              asn + 166    26       1.4 × 10.sup.-4                                                                 2 × 10.sup.5                              arg + 166    19       6.2 × 10.sup.-5                                                                 3 × 10.sup.5                              val + 166    1        1.4 × 10.sup. -4                                                                1 × 10.sup.4                      BVGRpN  Wild Type    2        1.1 × 10.sup.-3                                                                 2 × 10.sup.3                              asp + 166    2        4.1 × 10.sup.-5                                                                 5 × 10.sup.4                              glu + 166    2        2.7 × 10.sup.-5                                                                 7 × 10.sup.4                              asn + 166    1        1.2 × 10.sup.-4                                                                 8 × 10.sup.3                      ______________________________________                                    

The Kcat/Km ratio for each of the mutants varied from that of thewild-type enzyme. As a measure of catalytic efficiency, these ratiosdemonstrate that enzymes having much higher activity against a givensubstrate can be readily designed and selected by screening inaccordance with the invention herein. For example, A166 exhibits over 2times the activity of the wild type on sAAPFpN.

This data also demonstrates changes in substrate specificity uponmutation of the wild type enzyme. For example, the Kcat/Km ratio for theD166 and E166 mutants is higher than the wild type enzyme with the BVGpNsubstrate, but qualitatively opposite results were obtained uponincubation with sAAPFpN. Accordingly, the D166 and E166 mutants wererelatively more specific for BVGRpN than for sAAPFpN.

EXAMPLE 19 Mutant Subtilisin Exhibiting Modified pH-Activity Profile

The pH profile of the Cys+222 mutant obtained in Example 16 was comparedto that of the wild type enzyme. 10 μl of 60 mg/ml sAAPFpN in DMSO, 10μl of Cys+222 (0.18 mg/ml) or wild type (0.5 mg/ml) and 980 μl of buffer(for measurements at pH 6.6, 7.0 and 7.6, 0.1M NaPO₄ buffer; at pH 8.2,8.6 and 9.2, 0.1M tris buffer; and at pH 9.6 and 10.0, 0.1M glycinebuffer), after which the initial rate of change in absorbance at 410 nmper minute was measured at each pH and the data plotted in FIG. 15. TheCys+222 mutant exhibits a sharper pH optimum than the wild type enzyme.

EXAMPLE 20 Site-Specific Mutagenesis of the Subtilisin Gene at Position169

The procedure of Examples 13-14 was followed in substantial detail,except that the mutagenesis primer differed (the primer shown in FIG. 16was used), the two restriction enzymes were KpnI and EcoRV rather thanPstI and KpnI and the resulting constructions differed, as shown in FIG.16.

Bacillus strains excreting mutant subtilisins at position 169 wereobtained as described below in Example 16. The mutant subtilisinsexhibiting substitutions of ala and set for the wild-type residue wererecovered and assayed for changes in kinetic features. The assayemployed SAAPFpN at pH 8.6 in the same fashion as set forth in Example18. The results were as follows:

                  TABLE E                                                         ______________________________________                                        Enzyme    Kcat (s.sup.-1)                                                                            Km(M)     Kcat/Km                                      ______________________________________                                        ala + 169 58           7.5 × 10.sup.-5                                                                   8 × 10.sup.5                           ser + 169 38           8.5 × 10.sup.-5                                                                   4 × 10.sup.5                           ______________________________________                                    

EXAMPLE 21 Alterations in Specific Activity on a Protein Substrate

Position 166 mutants from Examples 15 and 16 were assayed for alterationof specific activity on a naturally occuring protein substrate. Becausethese mutant proteases could display altered specificity as well asaltered specific activity, the substrate should contain sufficientdifferent cleavage sites i.e., acidic, basic, neutral, and hydrophobic,so as not to bias the assay toward a protease with one type ofspecificity. The substrate should also contain no derivitized residuesthat result in the masking of certain cleavage sites. The widely usedsubstrates such as hemoglobin, azocollogen, azocasein, dimethyl casein,etc., were rejected on this basis. Bovine casein, α and α₂ chains, waschosen as a suitable substrate.

A 1 percent casein (w/v) solution was prepared in a 100 mM Tris buffer,pH 8.0, 10 mM EDTA. The assay protocol is as follows:

790 μl 50 mM Tris pH 8.2

100 μl 1 percent casein (Sigma) solution

10 μl test enzyme (10-200 μg).

This assay mixture was mixed and allowed to incubate at room temperaturefor 20 minutes. The reaction was terminated upon the addition of 100 μl100 percent trichloroacetic acid, followed by incubation for 15 minutesat room temperature. The precipitated protein was pelleted bycentrifugation and the optical density of the supernatant was determinedspectrophotometrically at 280 nm. The optical density is a reflection ofthe amount of unprecipitated, i.e., hydrolyzed, casein in the reactionmixture. The amount of casein hydrolysed by each mutant protease wascompared to a series of standards containing various amounts of the wildtype protease, and the activity is expressed as a percentage of thecorresponding wild type activity. Enzyme activities were converted tospecific activity by dividing the casein hydrolysis activity by the 280nm absorbance of the enzyme solution used in the assay.

All of the mutants which were assayed showed less specific activity oncasein than the wild type with the exception of Asn+166 which was 26percent more active on casein than the wild type. The mutant showing theleast specific activity was ile+166 at 0.184 of the wild type activity.

We claim:
 1. A method for making a mutant subtilisin, the methodcomprising the steps:a) obtaining a DNA fragment comprising a regionencoding a Bacillus subtilisin or subtilisin precursor; b) introducing amutation into said DNA which, upon expression of said DNA, results inthe substitution of at least one amino acid, said mutation beingintroduced into the subtilisin at one or more amino acid positions inthe subtilisin equivalent to tyr-1, asp+32, asn+155, tyr+104, met+222,gly+166, his+64, ser+221, ser+33, phe+189, tyr+217, ala+152, glu+156 orgly+169, in the corresponding amino acid sequence of the maturesubtilisin naturally produced by Bacillus amyloliquefaciens; c)transforming a suitable Bacillus host cell with said mutated DNA of stepb); d) recovering a mutant subtilisin expressed by the host celltransformed with said mutated DNA; and e) screening said mutantsubtilisin recovered in step d) for alterations of the enzymecharacteristics of substrate specificity, oxidative stability,pH-activity profile, or the rate of formation of mature subtilisin froma subtilisin precursor.
 2. The method of claim 1 wherein said subtilisinis prosubtilisin or preprosubtilisin.
 3. The method of claim 2 whereinsaid mutation is introduced into the prosubtilisin at position tyr-1. 4.The method of claim 1 wherein mutation is expressed as the substitutionof no more than one amino acid.
 5. A method for making a mutant Bacillussubtilisin, the method comprising the steps:a) obtaining a DNA fragmentcomprising a region encoding a Bacillus subtilisin or subtilisinprecursor; b) introducing a mutation into said DNA fragment within acodon region encoding an amino acid of the enzyme active site, a codonregion encoding an amino acid forming the primary substrate bindingsubsite or within a codon region encoding an amino acid present in theprimary sequence of the precursor that is involved in the processing ofsaid precursor into a mature subtilisin; c) transforming a suitableBacillus host cell with said mutated DNA; d) expressing said mutated DNAas a mutant of said subtilisin; e) recovering said mutant subtilisin;and f) screening said mutant subtilisin for alterations of the enzymecharacteristics of substrate specificity, oxidative stability,pH-activity profile, or the rate of formation of mature subtilisin froma subtilisin precursor.
 6. The method of claim 5 wherein said mutationis expressed as a change in no more than one amino acid of the precursorsubtilisin enzyme.
 7. The method of claim 5 wherein the mutation iswithin the enzyme active site.